Enveloped viruses must fuse viral and cellular membranes to transfer the viral nucleic acid into the host cell and initiate the infectious cycle. These viruses have evolved dedicated fusion proteins that catalyze this energetically unfavorable process. These fusion proteins fall into three classes as exemplified by influenza hemagglutinin (class I), flavivirus envelope proteins (class II) and rhabdovirus glycoproteins (class III). In response to specific triggers, these fusion proteins undergo dramatic conformational changes that bring the viral and target membranes into close proximity, lowering the energy barrier to membrane fusion. The mechanism by which class III proteins accomplish this is the least well understood. Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae, is the ideal model to study how class III fusion machines function as the structure of its single attachment and fusion glycoprotein was recently solved by X-ray crystallography in both pre and post fusion forms. Our long term objectives are to understand how VSV delivers its 286 MDa ribonucleoprotein core into cells to initiate the process of infection. Membrane fusion is a central step of this process. Here we have capitalized on the facile genetics of VSV and its robust growth in cell culture to develop new technologies to study the process of membrane fusion and viral entry. Our underlying hypothesis is that specific pH triggered conformational transitions in G drive the initial steps of membrane fusion, but that interactions between multiple G protein trimers are required to accomplish delivery of the ribonucleoprotein core of the virus across the membrane. We will examine this hypothesis in three interrelated aims. In specific aim 1, we will use genetic and biochemical approaches to determine the requirements in G for pH triggered conformational change, membrane fusion and viral infectivity. In specific aim 2, we will use high-resolution single particle imaging approaches to probe the relationships between hemifusion, fusion pore formation and transfer of the RNP across a lipid bilayer in vitro. In specific aim 3, we will use high resolution single particle imaging to determine the site of membrane fusion and RNP release in cells. Completion of these studies will reveal how a class III fusion protein functions to accomplish delivery of the viral contents into the cell. Consequently, these studies will provide new mechanistic insights into the process of enveloped virus membrane fusion and endocytic transport.